The determination of analytes is a widespread concern, especially in clinical diagnostics. Recently methods which comprise immunological reaction steps have, above all, been applied for this because of the great accuracy which can be achieved, as well as their wide applicability. Because of the advantage of easy handling, such methods are increasingly being carried out with the aid of a component of an immunological reaction bound to a solid phase. These methods allow, e.g., the use of test strips on which the entire reaction sequence proceeds solely by bringing the sample into contact with the strip.
Immunological methods of determination can be classified according to the nature of the participating reaction partners. One of these is the so-called competitive displacement test. In this procedure a bound, immobilized antigen, to which a labelled antibody is itself bound, is brought into contact with the sample. In the presence of analyte in the sample the immobilized analyte is displaced from the immunocomplex of immobilized antigen and labelled antibody by the analyte in the sample. The previously immobilized labelled antibody thus passes to the liquid phase, forms a complex with the analyte to be determined, and can be determined by means of its label after separating the liquid phase from the solid phase. The concentration of the analyte in the sample can be determined from this.
Such an immunotest is described for example in EP-A-0173375. A labelled Fab fragment is used as the labelled antibody which is present at the beginning of the test in a complex with the immobilized analyte.
A similar method is described in U.S. Pat. No. 4,436,236; however, an immobilized antigen is used which has a lower affinity to the labelled antibody than the analyte. Labelled antibody fragments are also preferably used in this method.
The methods of EP-A-0173375 and U.S. Pat. No. 4,436,236 are disadvantageous in that they exhibit high blank values for the measurement even if no analyte is in the sample; the magnitude of the signal is, however, relatively small.
An immunoassay is described in U.S. Pat. No. 4,277,560 in which a labelled analyte is bound reversibly to a solid phase via an immobilized antibody. The labelled analyte is displaced from the solid phase by the analyte present in the sample and can be used subsequently as a measure for the amount of analyte to be determined. This method is disadvantageous in that the accuracy of the results depends very strongly on the uniformity of the coating of the solid phase. Adequate accuracy is only achievable with difficulty.